Thrombospondin-1 type 1 repeat polypeptides

ABSTRACT

Thrombospondin-1 (TSP-1) is a potent inhibitor of tumor growth and angiogenesis. To elucidate the molecular mechanisms that are involved in the inhibition of tumor growth by the type 1 repeats (TSRs), recombinant versions of these motifs have been produced and have been assayed for their ability to inhibit the growth of experimental B16F10 and Lewis lung carcinomas. Recombinant proteins that contain all three TSRs (3TSR) or the second TSR with (TSR2+RFK) or without (TSR2) the transforming growth factor beta (TGFβ) activating sequence (RFK) have been expressed in  Drosophila  S2 cells. A recombinant protein containing all three type 1 repeats of TSP-2 and a recombinant protein containing the second TSR with the RFK sequence altered to QFK have also been produced. The data indicate that the TSRs inhibit tumor growth by inhibition of angiogenesis and regulation of tumor cell growth and apoptosis. The regulation of tumor cell growth and apoptosis is RFK-dependent while the inhibition of angiogenesis is not. The invention relates to polypeptides based on the amino acid sequence of human TSP-1 type 1 repeats. The polypeptides, variants, fragments and mutants thereof can be made by recombinant methods or can be made by chemical synthesis. The polypeptides can be formulated into pharmaceutical compositions and used in methods of therapy to reduce tumor growth.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/207,994 filed May 26, 2000, the entire teachings of which areincorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant HL28749from the National Heart, Lung and Blood Institute of the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Thrombospondin-1 is a potent inhibitor of angiogenesis (for a review,see Dawson, D. W. and Bouck, N. P., “Thrombospondin as an inhibitor ofangiogenesis,” In: B. A. Teicher (eds.), Antiangiogenic Agents in CancerTherapy, pp. 185–203, Totowa, N.J.; Humana Press, Inc., 1999). Itinhibits endothelial cell growth, migration and tube formation in vitro(Panetti, T. S., et al., J. Lab. Clin. Med., 129: 208–216, 1997). Invitro assays have shown that platelet thrombospondin-1 is involved inthrombosis, fibrinolysis, wound healing, inflammation, tumor cellmetastasis and angiogenesis. The major form of thrombospondin secretedby platelets and endothelial cells is TSP-1. Thrombospondin-1 (TSP-1) isan angiogenesis inhibitor. Thrombospondin-1 has three copies of the TSR.TSP-1 is a trimeric molecule. Thus, the fully assembled protein containsnine TSRs.

The ingrowth of new capillary networks into developing tumors isessential for the progression of cancer. As pointed out in a review byFolkman (Folkman, J., Proc. Natl. Acad. Sci. USA 95: 9064–9066, 1998),antiangiogenic therapy has little toxicity, does not require thetherapeutic agent to enter tumor cells or cross the blood-brain barrier,controls tumor growth independently of growth of tumor cellheterogeneity, and does not induce drug resistance. Thus, thedevelopment of pharmaceuticals that inhibit the process of angiogenesisor can inhibit abnormal cell proliferation by other mechanisms is animportant therapeutic goal.

SUMMARY OF THE INVENTION

The invention involves the use of a recombinant or synthetic version ora segment of the type 1 repeat domain (TSR) of human thrombospondin-1(TSP-1) or variants, fragments or mutants thereof to inhibit tumorgrowth by angiogenic or other activity. In particular, good anti-tumoractivity is obtained if the protein or protein fragment employedincludes the KRFK sequence (SEQ ID NO: 28) from the C-terminal end ofthe first TSR and a sufficient portion of the N-terminal end of thesecond TSR (through the first disulfide bond) to give the protein orprotein fragment tertiary structure similar to the wild-type TSRs. AcDNA encoding amino acids 411 through 473 of human TSP-1 (SEQ ID NO: 22)has been cloned into the pMT/BiP/V5His A expression vector. Therecombinant protein has been expressed and purified by standardprotocols. This protein is a potent inhibitor of tumor growth in mice.The growth of B16F10 melanoma cells and Lewis lung carcinoma cells isinhibited by 86% and 77%, respectively, with a single dailyinterperitonial dose of 2.5 mg/kg/day. The inclusion of the sequenceKRFK (SEQ ID NO: 28) at the N-terminus of the recombinant proteinsignificantly increased the anti-tumor activity of this protein. Theprotein that contains the KRFK sequence (SEQ ID NO: 28) is termedbroadly herein a polypeptide comprising the second type 1 repeat ofhuman TSP-1, but not comprising the RFK sequence, or the other domainsof TSP-1; also, a “TSR2+RFK polypeptide or protein.” At 1 mg/kg/day theTSR2+RFK protein described in the Examples inhibited B16F10 tumor growthby 80%. The protein that lacks the KRFK sequence is termed broadlyherein a polypeptide comprising the second type 1 repeat of human TSP-1,and the RFK sequence, but not the other domains of TSP-1; also, a “TSR2polypeptide or protein” The TSR2 protein described in the Examplesinhibited tumor growth by 38%. This result is contrary to previouslypublished results using synthetic peptides and an orthotopic model ofbreast cancer, in that the KRFK sequence (SEQ ID NO: 28) was previouslyreported to lack anti-tumor activity.

The mechanism of action for the inhibition of tumor growth by a TSR2+RFKprotein involves inhibition of angiogenesis, induction of tumor cellapoptosis and inhibition of tumor cell proliferation. The latter twoactivities appear to require the KRFK sequence (SEQ ID NO: 28) in thatthese effects are not observed with the TSR2 protein. The KRFK sequence(SEQ ID NO: 28) of TSP-1 has not been reported to induce tumor cellapoptosis previously.

The invention relates to polypeptides based on the amino acid sequenceof human thrombospondin 1, and more specifically relates to: (1) apolypeptide comprising the three type 1 repeats of human TSP-1, but notcomprising the other domains of TSP-1 (a 3TSR or 3TSR (TSP1) polypeptideor protein); (2) a polypeptide comprising the second type 1 repeat ofhuman TSP-1, but not comprising the RFK sequence or the other domains ofTSP-1 (a TSR2 polypeptide or protein); (3) a polypeptide comprising thesecond type 1 repeat of human TSP-1 and the RFK sequence, but not theother domains of TSP-1 (a TSR2+RFK polypeptide or protein); (4) apolypeptide comprising the second type 1 repeat of human TSP-1 and theRFK sequence altered to QFK, but not the other domains of TSP-1 (aTSR2+QFK polypeptide or protein), and (5) a polypeptide comprising allthree type 1 repeats of human TSP-2, but not the other domains of TSP-2[a 3TSR (TSP2) polypeptide or protein]. See, for the domains of TSP-1,Table 1.

Further embodiments are fragments, variants and mutants of thesepolypeptides having amino acid sequences that differ from the type 1repeat segment of the human TSP-1 or human TSP-2. Variants can have, forexample, flanking amino acid sequence, amino acid substitutions,deletions, or additions, or some combination. The polypeptides,variants, fragments and mutants can be made by recombinant methods orcan be made by chemical synthesis, using L-amino acids, analogues orderivatives thereof, or D-amino acids or related compounds, or they canbe made of only L-amino acids.

Also described herein are nucleic acid molecules involved in the makingof constructs encoding the polypeptides described herein, vectors, andhost cells that can be used in recombinant methods of producing thepolypeptides.

The polypeptides can be formulated into pharmaceutical compositions andused in methods of therapy to reduce tumor growth. For example, aTSR2+RFK polypeptide, or a conservative variant thereof, can be combinedwith a pharmaceutically acceptable carrier. A fragment of the TSR2+RFKwith up to 30 consecutive amino acid residues deleted relative to theTSR2+RFK polypeptide having SEQ ID NO: 22 can also be formulated into apharmaceutical composition. Further, variants of the TSR2+RFKpolypeptide, especially conservative variants, and variants at least70%, 80%, 90% or 95% identical in amino acid sequence to TSR2+RFK can beformulated with a pharmaceutically acceptable carrier.

The TSR2+RFK polypeptides, fragments, variants and mutants thereof, aswell as other polypeptides described herein, can be administered to ahuman patient in an amount sufficient to induce apoptosis, inhibitneovascularization, or otherwise inhibit growth in cancerous cells(e.g., cells within a tumor).

A further embodiment of the invention is a method of killing cancerouscells in a patient, comprising administering to said patient an amountof a TSR2+RFK polypeptide fragment sufficient to induce apoptosis insaid cancerous cells, wherein said fragment has at least about the sameapoptotic and anti-angiogenic activity as TSR2+RFK as illustrated in theExamples, and has up to 30 consecutive amino acids deleted from thecarboxy terminus of TSR2+RFK. In variations of the method, a variantwhich is at least 70%, 80%, 90% or 95% identical in amino acid sequenceto TSR2+RFK is administered to a patient, wherein the variant can becombined with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing TGFβ activation by the TSR2+RFK protein ofthe Examples. Saline, the TSR2 or the TSR2+RFK recombinant proteins (5.0μg/ml) were incubated with B16F10 cells overnight in serum-free DMEM.The levels of active and total TGFβ were determined using the MLEC assay(Abe, M., et al., Anal. Biochem, 216:276–84, 1994). The asteriskindicates p<0.005 relative to control.

FIGS. 2A and 2B are graphs showing the effect of TSP-1 and theTSR-containing recombinant proteins of the Examples on endothelial cellmigration. HDMEC migration in the presence of varying concentrations ofTSP-1 (FIG. 2A, open squares), 3TSR (FIG. 2A, filled diamonds), TSR2(FIG. 2B, open squares) or TSR2+RFK (FIG. 2B, filled diamonds) wasdetermined after four hours. Migration in response to FGF-2 in theabsence of inhibitors was considered 100% migration and migration in theabsence of FGF-2 was considered 0% migration.

FIGS. 3A and 3B are graphs showing growth of B16F10 tumors in micetreated with platelet TSP-1 (FIG. 3A) or 3TSR protein (FIG. 3B). Eachtreatment group contained six mice. Treatment was initiated four daysafter subcutaneous injection of 1×10⁶ tumor cells. The mice received oneIP injection each day of saline (open squares), or 0.25 mg/kg/day (opencircles), 1.0 mg/kg/day (filled triangles) or 2.5 mg/kg/day (filledcircles) of 3TSR protein as described in the Examples. Tumor volume wasdetermined every other day with a dial caliper. The mean and standarderror are plotted.

FIG. 4 is a graph showing growth of Lewis lung carcinoma in mice treatedwith recombinant proteins. Each treatment group contained six mice.Treatment was initiated four days after subcutaneous injections of 1×10⁶tumor cells. The mice received an IP injection each day of saline(filled squares), or the 3TSR protein (filled circles) or the TSR2+RFKprotein (filled triangles). The recombinant proteins were used at a doseof 2.5 mg/kg/day. Tumor volume was determined every other day with dialcaliper. The mean and standard error are plotted.

FIGS. 5A and 5B are graphs showing the growth of B16F10 tumors in micetreated with TSR2+RFK protein (FIG. 5A) or TSR2 protein (FIG. 5B). Eachtreatment group contained six mice. Treatment was initiated four daysafter subcutaneous injection of 1×10⁶ tumor cells. The mice received oneIP injection each day of saline (open squares), or 0.25 mg/kg/day (opencircles), 1.0 mg/kg/day (filled triangles) or 2.5 mg/kg/day (filledcircles) of recombinant protein. Tumor volume was determined every otherday with a dial caliper. The mean and standard error are plotted.

FIGS. 6A, 6B and 6C are bar graphs showing the quantitation of bloodvessels (FIG. 6A), tumor cell apoptosis (FIG. 6B), and tumor cellproliferation (FIG. 6C). Tumors from mice treated with saline (solidbars), 1.0 mg/kg/day of TSR2+RFK protein (hatched bars) or 1.0 mg/kg/dayof TSR2 protein (shaded bars) were fixed and tissue was prepared forhistology. The asterisk indicates p<0.005 as compared to control in allcases except the level of apoptosis induced by TSR2 (FIG. 6B, shadedbar) where p≦0.025. A comparison of the vessel density for the micetreated with the TSR2+RFK (FIG. 6A, hatched bar) or TSR2 (FIG. 6A,shaded bar) yields p≦0.05.

FIG. 7 is a representation of the amino acid sequence of human TSP-1(SEQ ID NO: 1). The type 1 repeats of TSP-1 are, as illustrated here, 1)amino acids 361–416; 2) amino acids 417–473; and 3) amino acids 474–530.Also shown in FIG. 7 are the complete type 2 repeats, the type 3repeats, and the procollagen homology region.

FIG. 8 is a bar graph showing the effect of treatment with recombinant,TSP-1 peptides or controls on the tumor volume from melanoma B16F10 inC57BL/6 mice.

FIG. 9 is a bar graph showing the effect of treatment with recombinantTSP peptides or saline as control on the tumor volume from melanomaB16F10 in C57Bl/6 mice.

FIG. 10 is a representation of the amino acid sequence of mousethrombospondin 2, including signal sequence (SEQ ID NO: 24). SeeNational Center for Biotechnology Information Accession No. NP 035711,PID g6755779, as in Laherty, C. D. et al., J. Biol. Chem.267(5):3274–3281, 1992.

FIG. 11 is a representation of the amino acid sequence of humanthrombospondin 2, including signal sequence (SEQ ID NO: 26). SeeNational Center for Biotechnology Information Accession No. TSHUP2, PIDg1070641, as in LaBell, T. L. et al., Genomics 12(3):421–429, 1992.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The data presented here show that administration of recombinant proteins(also herein: “polypeptides”) that contain the TSRs of TSP-1 or the TSRsof TSP-2 inhibit the growth of experimental tumors. Inhibition has beenobserved with both B16F10 melanoma and Lewis lung carcinoma cell lines.The 3TSR and TSR2+RFK proteins are relatively potent, showing 70%–80%inhibition of tumor volume at 2.5 mg/kg/day. This concentration is100–300 nmoles/kg/day. Injection with intact TSP-1 protein at 0.25mg/kg/day (0.6 nmoles/kg/day) resulted in a 56% reduction of tumorgrowth, indicating that the intact protein is considerably more activethan the recombinant proteins at low doses.

Several activities of the TSRs may contribute to the inhibition of tumorgrowth. These include the ability to (1) inhibit angiogenesis, (2)reduce tumor cell proliferation, (3) activate TGFβ, and (4) regulateextracellular proteases. The results described here show that systemictreatment with the TSRs of TSP-1 or TSP-2 reduces vessel density intumors.

The rate of tumor cell apoptosis is a key factor in the determination ofthe rate of tumor growth (Parangi, P., et al., Proc. Natl. Acad. Sci.(USA), 93: 2002–2007, 1996; Naik, P. N., et al., Genes & Dev.,10:2105–2116, 1996). Described herein are results showing that TSP-1proteins that include the RFK sequence significantly increase the rateof tumor cell apoptosis. Whereas TSP-1 has been reported to inhibittumor cell proliferation and induce endothelial cell apoptosis, thisstudy demonstrates that the TSR-containing proteins induce tumor cellapoptosis (Guo, NH., et al., Cancer Res., 57:1735–1742, 1997; Guo,N.-H., et al., Cancer Res., 58: 3154–3162, 1998). This activity isenhanced by the addition of the DKRFK (SEQ ID NO: 2) sequence at theNH₂-terminus, because the TSR2 protein did not increase the level ofapoptosis of the tumor cells to the same extent as the TSR2+RFK protein.An increase in tumor cell apoptosis is frequently associated withanti-angiogenic therapy due to the decrease in nutrients that isassociated with the decrease in blood supply. These data herein indicatethat increased tumor cell apoptosis occurs as a direct result of theTSR2+RFK protein treatment rather than indirectly through inhibition ofangiogenesis. Whereas, the TSR2+RFK and TSR2 proteins reduce vesseldensity to comparable levels, the TSR2+RFK protein has a more profoundeffect on tumor cell apoptosis. In addition, this protein significantlydecreases tumor cell proliferation in vivo.

The data presented here indicate that inclusion of the additionalsequence DKRFK (SEQ ID NO: 2) at the N-terminus of the TSR2 protein toyield the TSR2+RFK protein significantly increases the anti-tumoractivity of the protein. The difference in activity of the protein thatcontains the DKRFK (SEQ ID NO: 2) sequence as compared to the one thatdoes not may be due to the activation of TGFβ, an increased affinity forheparan sulfate proteoglycans or an unidentified activity of thissequence. The additional sequence in the TSR2+RFK protein includes theRFK sequence that reportedly activates TGFβ (Schultz-Cherry, S., et al.,J. Biol. Chem., 270: 7304–7310, 1995). In experiments using the culturesupernatant from B16F10 cells, it was shown that the TSR2+RFK proteinactivates TGFβ, while the TSR2 protein does not. TGFβ has pleiotropiceffects on tumor growth. At early stages of tumorigenesis, TGFβ may actas a tumor suppressor gene (Engle, S. J., et al., Cancer Research, 59:3379–3386, 1999; Tang, B., et al., Nature Med., 4: 802–807, 1998). TGFβcan induce apoptosis of several different tumor cell lines (Guo, Y. andKypianou, N., Cancer Research, 59: 1366–1371, 1999 and referencestherein). At later stages of tumor growth, TGFβ can stimulateangiogenesis through the recruitment of inflammatory and stromal cells(Pepper, M., Cytokine & Growth Factor Reviews, 8: 21–43, 1997).

The data presented here indicate that recombinant proteins that includethe TSRs have therapeutic value as inhibitors of tumor growth. They actto inhibit angiogenesis and to induce tumor cell apoptosis. Since thesemechanisms of action are probably different from other inhibitors ofneoplasia, a combinational approach that includes TSRs with otherinhibitors of tumor growth can be an effective treatment for cancer.

The RFK sequence mediates the ability of TSP-1 to activate TGFβ(Schultz-Cherry, S. et al., J. Biol. Chem. 270:7304–7310, 1995).Mutation of the arginine residue (R) to glutamine (Q) abolishes theability of synthetic peptides to activate TGFβ. A recombinant version ofthe TSR2+RFK protein was produced in which Q is substituted for R. Thisprotein is designated TSR2+QFK. In the B16F10 experimental tumor model,TSR2+QFK is less effective as an inhibitor of tumor growth than TSR2+RFK(FIG. 8). The activity of TSR2+QFK is comparable to that of TSR2. Arecombinant version of all three type 1 repeats of mouse TSP-2 has alsobeen prepared. TSP2 has the sequence RIR in the position where TSP-1 hasRFK, and has been reported to be unable to activate TFGβ(Schultz-Cherry, S. et al, J. Biol. Chem. 270:7304–7310, 1995). Therecombinant protein that includes all three TSRs of mouse TSP-2 issignificantly less potent as an inhibitor of B16F10 tumor growth thanall three type 1 repeats of human TSP-1 (FIG. 9).

The ability of TSR2+RFK to inhibit tumor growth was also evaluated inthe presence of a soluble form of the TGFβ receptor. Systemic injectionof this reagent has been shown to inhibit endogenous TGFβ activity(Smith, J. D. et al., Circ. Res. 84:1212–22, 1999). Concurrent treatmentof B16F10 tumor-bearing mice with TSR2+RFK and the soluble form of theTGFβ receptor resulted in a significant loss of anti-tumor activity(FIG. 8). The level of inhibition of tumor growth under thesecircumstances was comparable to that observed with the TSR2 or TSR2+QFKproteins. Animals that received control injections of saline with thesoluble TGFβ receptor produced tumors that are about 15% larger thanthose produced in the mice that received only saline. This suggests thatTGFβ that is activated by endogenous TSP-1 or by other means is normallysuppressing tumor growth.

Taken together, these data indicate that the ability of the TSR2+RFKprotein to inhibit tumor growth is due in part to the activation of TGFβby the RFK sequence. This conclusion is consistent with the observationthat both TSR2+RFK and TGFβ inhibit B16F10 tumor cell proliferation invitro.

The recombinant protein is more effective than the intact TSP-1 proteinin inhibiting tumor growth. The recombinant protein includes specificactive sequences and does not include other domains of TSP-2 or TSP-1that may decrease the over all activity. See Table 1 for activesequences of TSP-1 (taken from chapter 2, “The Primary Structure of theThrombospondins” In The Thrombospondin Gene Family (J. C. Adams et al.,eds.) Springer-Verlag, Heidelberg (1995. The recombinant protein can bemade in large quantities and does not require human tissue as a sourceof protein. This protein is folded like the wild-type protein and can bemore stable in circulation than peptides. It can also be modified toinclude sequences that will target it to tumor tissue. Because theseproteins are derived from portions of human proteins, they should not beimmunogenic in humans.

TABLE 1 Active Regions of Interest Within Thrombospondin-1 DomainSequence Function Procollagen NGVQYRN (SEQ ID NO: 4) Anti-angiogenesishomology Type 1 CSVTCG (SEQ ID NO: 5) Cell binding repeats WSXWSXW (SEQID NO: 6) Heparin binding GGWSHW (SEQ ID NO: 7) TGF-β and Fibronectinbinding RFK TGF-β activation SPWDICSVTCGGGVQKRSR Anti-angiogenesis (SEQID NO: 8) Type 2 DVDEC(X)₆C(X)₈CENTDPGYNCLPC Calcium binding repeats(SEQ ID NO: 9)

In one aspect, the invention comprises polynucleotides or nucleic acidmolecules that encode polypeptides whose amino acid sequences arederived from human TSP-1. By the genomic structure, the type 1 repeatsof TSP-1 are amino acid residues 359–414 (first), amino acid residues415–473 (second), and 474–531 (third). In another case, the polypeptidesencoded by the polynucleotides of the invention are fragments orvariants of the immediately aforementioned polypeptides, which haveactivity that is similar in quality and quantity (for example, plus orminus one order of magnitude in an assay) to the anti-angiogenic and/orapoptosis increasing and/or anti-tumor growth activity of thepolypeptides tested in the Examples. A fragment of a TSR2+RFK, TSR2+QFK,or TSR2 polypeptide can, for instance, have up to 30 (any number from 1to 30) consecutive amino acid residues deleted from the carboxyterminus, relative to SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 21,respectively. The carboxy terminal deletion in a fragment of apolypeptide can be, for example, 5, 10, 15, 20 or 25 consecutive aminoacids.

The polynucleotides that encode the polypeptides described herein can bemade by recombinant methods, can be made synthetically, can bereplicated by enzymes in in vitro (e.g., PCR) or in vivo systems (e.g.,by suitable host cells, when inserted into a vector appropriate forreplication within the host cells), or can be made by a combination ofmethods. The polynucleotides of the invention can include DNA and itsRNA counterpart.

As used herein, “nucleic acid,” “nucleic acid molecule,”“oligonucleotide” and “polynucleotide” include DNA and RNA and chemicalderivatives thereof, including phosphorothioate derivatives and RNA andDNA molecules having a radioactive isotope or a chemical adduct such asa fluorophore, chromophore or biotin (which can be referred to as a“label”). The RNA counterpart of a DNA is a polymer of ribonucleotideunits, wherein the nucleotide sequence can be depicted as having thebase U (uracil) at sites within a molecule where DNA has the base T(thymidine).

Isolated nucleic acid molecules or polynucleotides can be purified froma natural source or can be made recombinantly. Polynucleotides referredto herein as “isolated” are polynucleotides purified to a state beyondthat in which they exist in cells. They include polynucleotides obtainedby methods described herein, similar methods or other suitable methods,and also include essentially pure polynucleotides produced by chemicalsynthesis or by combinations of biological and chemical methods, andrecombinant polynucleotides that have been isolated. The term “isolated”as used herein for nucleic acid molecules, indicates that the moleculein question exists in a physical milieu distinct from that in which itoccurs in nature. For example, an isolated polynucleotide may besubstantially isolated with respect to the complex cellular milieu inwhich it naturally occurs, and may even be purified essentially tohomogeneity, for example as determined by agarose or polyacrylamide gelelectrophoresis or by A₂₆₀/A₂₈₀ measurements, but may also have furthercofactors or molecular stabilizers (for instance, buffers or salts)added.

The invention further relates to fusion proteins comprising, forexample: (1) a polypeptide comprising the three type 1 repeats of humanTSP-1, but not comprising the other domains of TSP-1; (2) a polypeptidecomprising the second type 1 repeat of human TSP-1, but not comprisingthe RFK sequence or the other domains of TSP-1; (3) a polypeptidecomprising the second type 1 repeat of human TSP-1 and the RFK sequence,but not the other domains of TSP-1; (4) a polypeptide comprising thesecond type 1 repeat of human TSP-1 and the RFK sequence altered to QFK,but not the other domains of TSP-1; or (5) a polypeptide comprising allthree type 1 repeats of human TSP-2, but not the other domains of TSP-2,wherein any one of (1), (2), (3), (4) or (5) is linked to a secondmoiety not occurring in TSP-1 or TSP-2 as found in nature. The secondmoiety can be an amino acid, peptide or polypeptide, and can haveenzymatic or binding activity of its own. The first moiety can be in anN-terminal location, C-terminal location or internal to the fusionprotein. The second moiety can comprise a linker sequence and anaffinity ligand, for example.

Variants of the polypeptide include those having amino acid sequencesdifferent from sequences which can be seen as contiguous portions of thesequence in FIG. 7 (which illustrates, for example, amino acid residues361–530 (SEQ ID NO: 20), 411–473 (SEQ ID NO: 22) and 416–473 (SEQ ID NO:21)). Variants can have, for instance, several, such as 5 to 10, 1 to 5,or 4, 3, 2 or 1 amino acids substituted, deleted, or added, in anycombination, compared to the sequences which are a portion of thesequence in FIG. 7. In one embodiment, variants have silentsubstitutions, additions and/or deletions that do not significantlyalter the properties and activities of the polypeptide. See Table 2 forexamples of conservative amino acid substitutions. Variants can also bemodified polypeptides in which one or more amino acid residues aremodified.

The invention also encompasses variant polypeptides having a lowerdegree of identity but having sufficient similarity so as to perform oneor more of the same functions performed by the polypeptides describedherein by amino acid sequence. Similarity for a polypeptide isdetermined by conserved amino acid substitution. Such substitutions arethose that substitute a given amino acid in a polypeptide by anotheramino acid of like characteristics. Conservative substitutions arelikely to be phenotypically silent. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr, exchange of the acidic residues Asp and Glu, substitutionbetween the amide residues Asn and Gln, exchange of the basic residuesLys and Arg and replacements among the aromatic residues Phe, Tyr andTrp. Guidance concerning which amino acid changes are likely to bephenotypically silent is found in Bowie et al., Science 247:1306–1310(1990). See also Table 2.

TABLE 2 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

“Sequence identity,” as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., two polynucleotides ortwo polypeptides. When a subunit position in both of the two moleculesis occupied by the same monomeric subunit, e.g., if a position in eachof two polypeptides is occupied by serine, then they are identical atthat position. The identity between two sequences is a direct functionof the number of matching or identical positions, e.g., if half (e.g., 5positions in a polymer 10 subunits in length) of the positions in twopeptide or compound sequences are identical, then the two sequences are50% identical; if 90% of the positions, e.g., 9 of 10 are matched, thetwo sequences share 90% sequence identity. By way of example, the aminoacid sequences R₂R₅R₇R₁₀R₆R₃ and R₉R₈R₁R₁₀R₆R₃ have 3 of 6 positions incommon, and therefore share 50% sequence identity, while the sequencesR₂R₅R₇R₁₀R₆R₃ and R₈R₁R₁₀R₆R₃ have 3 of 5 positions in common, andtherefore share 60% sequence identity. The identity between twosequences is a direct function of the number of matching or identicalpositions. Thus, if a portion of the reference sequence is deleted in aparticular peptide, that deleted section is not counted for purposes ofcalculating sequence identity, e.g., R₂R₅R₇R₁₀R₆R₃ and R₂R₅R₇R₁₀R₃ have5 out of 6 positions in common, and therefore share 83.3% sequenceidentity.

Identity is often measured using sequence analysis software e.g., BLASTNor BLASTP (available at website for National Center for BiotechnologyInformation, National Institutes of Health). The default parameters forcomparing two sequences (e.g., “Blast”-ing two sequences against eachother by BLASTN (for nucleotide sequences) are reward for match=1,penalty for mismatch=−2, open gap=5, extension gap=2. When using BLASTPfor protein sequences, the default parameters are reward for match=0,penalty for mismatch=0, open gap=11, and extension gap=1.

When two sequences share “sequence homology,” it is meant that the twosequences differ from each other only by conservative substitutions. Forpolypeptide sequences, such conservative substitutions consist ofsubstitution of one amino acid residue at a given position in thesequence for another amino acid residue of the same class (e.g., aminoacids that share characteristics of hydrophobicity, charge, pK or otherconformational or chemical properties, e.g., valine for leucine,arginine for lysine), or by one or more non-conservative amino acidsubstitutions, deletions, or insertions, located at positions of thesequence that do not alter the conformation or folding of thepolypeptide to the extent that the biological activity of thepolypeptide is destroyed. Examples of “conservative substitutions”include substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another; the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between threonine andserine; the substitution of one basic residue such as lysine, arginineor histidine for another; or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another; or the use of achemically derivatized residue in place of a non-derivatized residue;provided that the polypeptide displays the requisite biologicalactivity. Two sequences which share sequence homology may called“sequence homologs.”

Homology, for polypeptides, is typically measured using sequenceanalysis software (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Protein analysis softwarematches similar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid, asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine.

Embodiments of the invention include not only the polypeptidesconsisting of SEQ ID NOs 20, 21, 22, 23, 25 and 27, but also anypolypeptide which is at least 70% or 80%, more preferably 90%, and stillmore preferably, 95% identical to any of the TSP polypeptides identifiedherein by SEQ ID NO, wherein the polypeptide has at least about the sameanti-angiogenic or apoptotic or anti-tumor activity as the polypeptideidentified by SEQ ID NO. These activities can be determined by methodsdescribed herein, for example, the TUNEL assay for apoptosis (Abe, M. etal., Anal. Biochem. 216:276–84, 1994), the measurement of capillarydensity for anti-angiogenic effect, as described in Example 2, or thecounting of tumor cells to determine cell proliferation or theinhibition thereof. Other methods known in the art can also be used.

A further embodiment of the invention is a 3TSR, TSR2, TSR2+RFK orTSR2+QFK polypeptide, or an active variant or fragment of any of theforegoing, wherein the region of the polypeptide derived from the humanTSP-1 amino acid sequence is flanked on one or both ends by other aminoacid residues having an amino acid sequence which is not found as thehuman TSP-1 amino acid sequence that naturally occurs adjacent to theregion. Flanking peptide regions can be, for example, 1, 2, 3, 4 or 5,5–10, 10–20, or 20–30 amino acid residues in length.

Further embodiments of the invention are mutants of the 3TSR, TSR2,TSR2+RFK, TSR2+QFK, or 3TSR (TSP2) polypeptides. By “mutant” is meant apolypeptide that includes any change in the amino acid sequence relativeto the amino acid sequence of the polypeptide described herein by SEQ IDNO. Such changes can arise either spontaneously or by manipulations byman, by chemical energy (e.g., X-ray), or by other forms of chemicalmutagenesis, or by genetic engineering, or as a result of mating orother forms of exchange of genetic information. Mutations include, e.g.,base substitutions, deletions, insertions, inversions, translocations,or duplications. Mutant forms of the polypeptides can display eitherincreased or decreased anti-angiogenic activity and either increased ordecreased tumor growth inhibition activity relative to the polypeptidesidentified herein by SEQ ID NO. Such mutants may or may not alsocomprise additional amino acids derived from the process of cloning,e.g., amino acid residues or amino acid sequences corresponding to fullor partial linker sequences.

Mutants/fragments of the anti-angiogenic and/or anti-tumor polypeptidesof the present invention can be generated by PCR cloning. To make suchfragments, PCR primers can be designed from known nucleic acid sequencesin such a way that each set of primers will amplify a predicted regionof the coding sequence for the starting polypeptide. These amplifiedDNAs are then cloned into an appropriate expression vector, such as thepET22b vector, and the expressed polypeptide can ben tested for itsanti-angiogenic activity and anti-tumor activity. Alternatively, mutantsand fragments can be produced by chemical synthesis methods.

Proteins and polypeptides described herein can be assessed for theirangiogenic activity by using an assay such as the one described inTolsma, S. S. et al., J. Cell Biol. 122(2):497–511 (1993), an assaywhich measures the migration of bovine adrenal capillary endothelialcells in culture, or an assay which tests migration of cells into asponge containing an agent to be tested for activity. A further test forangiogenesis, which can also be adapted also to test anti-angiogenesisactivity, is described in Polverini, P. J. et al., Methods. Enzymol.198:440–450 (1991). More recently, methods to quantitate inhibition oftumor vascularization, and to quantitate inhibition of cornealneovascularization, were described in Blezinger, P. et al., NatureBiotechnology 17:343–348, 1999.

Another aspect of the invention relates to a method of producing apolypeptide of the invention, or a variant or fragment thereof, and toexpression systems and host cells containing a vector appropriate forexpression of a polypeptide of the invention. Cells that express such apolypeptide or a variant or a fragment thereof can be made andmaintained in culture, under conditions suitable for expression, toproduce protein for isolation. These cells can be procaryotic oreucaryotic. Examples of prokaryotic cells that can be used forexpression (as “host cells”; “cell” including herein cells of tissues,cell cultures, cell strains and cell lines) include Escherichia coli,Bacillus subtilis and other bacteria. Examples of eucaryotic cells thatcan be used for expression include yeasts such as Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris and other lowereukaryotic cells, and cells of higher eukaryotes such as those frominsects and mammals. Suitable cells of mammalian origin include primarycells, and cell lines such as CHO, HeLa, 3T3, BHK, COS, 293, and Jurkatcells. Suitable cells of insect origin include primary cells, and celllines such as SF9 and High five cells. (See, e.g., Ausubel, F. M. etal., eds. Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons Inc., (containing Supplements upthrough 2001)).

In one embodiment, host cells that produce a recombinant polypeptide,variant, or a fragment thereof can be made as follows. A gene encoding apolypeptide variant or fragment described herein can be inserted into anucleic acid vector, e.g., a DNA vector, such as a plasmid, virus orother suitable replicon (including vectors suitable for use in genetherapy, such as those derived from adenovirus or others; see, forexample Xu, M. et al., Molecular Genetics and Metabolism 63:103–109,1998) present in the cell as a single copy or multiple copies, or thegene can be integrated in a host cell chromosome. A suitable replicon orintegrated gene can contain all or part of the coding sequence for thepolypeptide, fragment or variant, operably linked to one or moreexpression control regions whereby the coding sequence is under thecontrol of transcription signals and linked to appropriate translationsignals to permit translation. The vector can be introduced into cellsby a method appropriate to the type of host cells (e.g., transformation,electroporation, infection). For expression from the gene, the hostcells can be maintained under appropriate conditions (e.g., in thepresence of inducer, normal growth conditions, etc.). Proteins orpolypeptides thus produced can be recovered (e.g., from the cells, theperiplasmic space, culture medium) using suitable techniques.

The invention also relates to isolated proteins or polypeptides.Isolated polypeptides or fragments or variants can be purified from anatural source or can be made recombinantly. Proteins or polypeptidesreferred to herein as “isolated” are proteins or polypeptides purifiedto a state beyond that in which they exist in cells and include proteinsor polypeptides obtained by methods described herein, similar methods orother suitable methods, and also include essentially pure proteins orpolypeptides, proteins or polypeptides produced by chemical synthesis orby combinations of biological and chemical methods, and recombinantproteins or polypeptides which are isolated. Thus, the term “isolated”as used herein, indicates that the polypeptide in question exists in aphysical milieu distinct from the cell in which its biosynthesis occurs.For example, an isolated polypeptide of the invention can be purifiedessentially to homogeneity, for example as determined by PAGE or columnchromatography (for example, HPLC), but may also have further cofactorsor molecular stabilizers added to the purified protein to enhanceactivity. In one embodiment, proteins or polypeptides are isolated to astate at least about 75% pure; more preferably at least about 85% pure,and still more preferably at least about 95% pure, as determined byCoomassie blue staining of proteins on SDS-polyacrylamide gels.

“Polypeptide” as used herein indicates a molecular chain of amino acidsand does not refer to a specific length of the product. Thus, peptides,oligopeptides and proteins are included within the definition ofpolypeptide. This term is also intended to include polypeptides thathave been subjected to post-expression modifications such as, forexample, glycosylations, acetylations, phosphorylations and the like. Inaddition, the polypeptides, fragments and variants of the invention canbe synthesized, by laboratory or by biosynthetic methods, using aminoacids that carry modifications such as glycosylation, acetylation,phosphorylation, or chemical adducts for the purpose of providing achromophoric, fluorophoric, radioactive, or biotin label, for example.

Fusion proteins comprising a polypeptide described herein can beproduced by a variety of methods. For example, a polypeptide can beproduced by the insertion of a TSP gene or portion thereof into asuitable expression vector, such as Bluescript SK +/− (Stratagene),pGEX-4T-2 (Pharmacia), pET-15b, pET-20b(+) or pET-24(+) (Novagen). Theresulting construct can be introduced into a suitable host cell forexpression. Upon expression, the polypeptide can be purified from a celllysate by means of a suitable affinity matrix (see e.g., CurrentProtocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 2, pp.16.4.1–16.7.8, containing supplements up through 2001).

Polypeptides of the invention can be recovered and purified from cellcultures by well-known methods. The recombinant polypeptide can bepurified by ammonium sulfate precipitation, heparin-Sepharose affinitychromatography, gel filtration chromatography and/or sucrose gradientultracentrifugation using standard techniques. Further methods that canbe used for purification of the polypeptide include ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and highperformance liquid chromatography. Known methods for refolding proteincan be used to regenerate active conformation if the polypeptide isdenatured during isolation or purification.

Also included in the inventions are compositions containing, as abiological ingredient, a polypeptide of the invention, or a variant,fragment or mutant thereof to inhibit angiogenesis and/or increaseapoptosis or inhibit tumor growth by other mechanisms in mammaliantissues, and use of such compositions in the treatment of diseases andconditions characterized by, or associated with, angiogenic activity ormisregulated growth. Such methods can involve administration by oral,topical, injection, implantation, sustained release, or other deliverymethods that bring one or more of the polypeptides in contact with cellswhose growth is to be inhibited.

The present invention includes a method of treating anangiogenesis-mediated disease or cancer with a therapeutically effectiveamount of one or more polypeptides having anti-angiogenic activity oranti-tumor activity as described herein. Angiogenesis-mediated diseasescan include, but are not limited to, cancers, solid tumors, tumormetastasis, benign tumors (e.g., hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas), rheumatoidarthritis, psoriasis, ocular angiogenic diseases (e.g., diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis), Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibroma,and wound granulation. Polypeptide antiangiogenic agents and/oranti-tumor agents can be used, for example, after surgery or radiationto prevent recurrence of metastases, in combination with conventionalchemotherapy, immunotherapy, or various types of gene therapy notnecessarily directed against angiogenesis.

“Cancer” means neoplastic growth, hyperplastic or proliferative growthor a pathological state of abnormal cellular development, and includessolid tumors, non-solid tumors, and any abnormal cellular proliferation,such as that seen in leukemia. As used herein, “cancer” also meansangiogenesis-dependent cancers and tumors, i.e., tumors that require fortheir growth (expansion in volume and/or mass) an increase in the numberand density of the blood vessels supplying them with blood. “Regression”refers to the reduction of tumor mass and size. As used herein, the term“therapeutically effective amount” means the total amount of each activecomponent of the composition or method that is sufficient to show ameaningful benefit to a treated human or other mammal, i.e., treatment,healing, prevention or amelioration of the relevant medical condition,or an increase in rate of treatment, healing, prevention or ameliorationof such conditions. More specifically, for example, a therapeuticallyeffective amount of an anti-angiogenic polypeptide can cause ameasurable reduction in the size or numbers of tumors, or in their rateof growth or multiplication, compared to untreated tumors. Other methodsof assessing a “therapeutically effective amount,” can include theresult that blood vessel formation is measurably reduced in treatedtissues compared to untreated tissues.

One or more anti-tumor polypeptides as described herein may be used incombination with other compositions and procedures for the treatment ofdiseases. For example, a tumor may be treated conventionally withsurgery, radiation, chemotherapy, or immunotherapy, combined withanti-angiogenic polypeptides, and then anti-tumor polypeptides may besubsequently administered to the patient to extend the dormancy ofmicrometastases and to stabilize and inhibit the growth of any residualprimary tumor.

The compositions may further contain other agents which either enhancethe activity of the protein or complement its activity or use intreatment, such as chemotherapeutic or radioactive agents. Suchadditional factors and/or agents may be included in the composition toproduce a synergistic effect with protein of the invention, or tominimize side effects. Additionally, the compositions of the presentinvention may be administered concurrently with other therapies, e.g.,in conjunction with a chemotherapy, immunotherapy or radiation therapyregimen.

The anti-tumor composition of the present invention may be a solid,liquid or aerosol and may be administered by any known route ofadministration. Examples of solid compositions include pills, creams,and implantable dosage units. The pills may be administered orally, thetherapeutic creams may be administered topically. The implantable dosageunit may be administered locally, for example at a tumor site, or may beimplanted for systemic release of the composition, for examplesubcutaneously. Examples of liquid compositions include formulationsadapted for injection subcutaneously, intravenously, intraarterially,and formulations for topical and intraocular administration. Examples ofaerosol formulation include inhaler formulation for administration tothe lungs.

One or more polypeptides described herein can be provided as isolatedand substantially purified polypeptides in pharmaceutically acceptableformulations (including aqueous or nonaqueous carriers or solvents)using formulation methods known to those of ordinary skill in the art.These formulations can be administered by standard routes. In general,the combinations can be administered by the topical, transdermal,intraperitoneal, intracranial, intracerebroventricular, intracerebral,intravaginal, intrauterine, oral, rectal or parenteral (e.g.,intravenous, intraspinal, subcutaneous or intramuscular) route. Inaddition, the polypeptides can be incorporated into biodegradablepolymers allowing for sustained release of the compound, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of a tumor, or implanted so that the polypeptidesare slowly released systemically. Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of polypeptidethrough cannulae to the site of interest, such as directly into a growthor into the vascular supply to that growth. The biodegradable polymersand their use are described, for example, in detail in Brem et al.(1991, J. Neurosurg. 74:441–446), which is hereby incorporated byreference in its entirety.

As used herein, the terms “pharmaceutically acceptable,” as it refers tocompositions, carriers, diluents and reagents, represents that thematerials are capable of administration to or upon a mammal with aminimum of undesirable physiological effects such as nausea, dizziness,gastric upset and the like. The preparation of a pharmacologicalcomposition that contains active ingredients dissolved or dispersedtherein is well understood in the art and need not be limited based onformulation. Typically, such compositions are prepared as injectableseither as liquid solutions or suspensions, however, solid forms suitablefor solution, or suspensions, in liquid prior to use can also beprepared. The preparation can also be emulsified, for example, inliposomes.

The dosage of the polypeptide of the present invention will depend onthe disease state or condition being treated and other clinical factorssuch as weight and condition of the human or animal and the route ofadministration of the compound. It is to be understood that the presentinvention has application for both human and veterinary use. The methodsof the present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time.

The present invention also encompasses gene therapy whereby apolynucleotide encoding one or more polypeptides or one or more variantsor fragments thereof, is introduced and regulated in a patient. Variousmethods of transferring or delivering DNA to cells for expression of thegene product protein, otherwise referred to as gene therapy, aredisclosed in Gene Transfer into Mammalian Somatic Cells in Vivo, N. Yang(1992) Crit. Rev. Biotechnol. 12(4):335–356, which is herebyincorporated by reference. Gene therapy encompasses incorporation of DNAsequences into somatic cells or germ line cells for use in either exvivo or in vivo therapy. Gene therapy can function to replace genes,augment normal or abnormal gene function, and to combat infectiousdiseases and other pathologies.

Strategies for treating these medical problems with gene therapy includetherapeutic strategies such as identifying the defective gene and thenadding a functional gene to either replace the function of the defectivegene or to augment a slightly functional gene; or prophylacticstrategies, such as adding a gene for the product protein that willtreat the condition or that will make the tissue or organ moresusceptible to a treatment regimen. For example, a gene encoding apolypeptide described herein can be inserted into tumor cells of apatient and thus inhibit angiogenesis.

Gene transfer methods for gene therapy fall into three broad categories:physical (e.g., electroporation, direct gene transfer and particlebombardment), chemical (e.g., lipid-based carriers, or other non-viralvectors) and biological (e.g., virus-derived vector and receptoruptake). For example, non-viral vectors may be used which includeliposomes coated with DNA. Such liposome/DNA complexes may be directlyinjected intravenously into the patient. It is believed that theliposome/DNA complexes are concentrated in the liver where they deliverthe DNA to macrophages and Kupffer cells. These cells are long lived andthus provide long term expression of the delivered DNA. Additionally,vectors or the “naked” DNA of the gene may be directly injected into thedesired organ, tissue or tumor for targeted delivery of the therapeuticDNA.

In vivo gene transfer involves introducing the DNA into the cells of thepatient when the cells are within the patient. Methods include usingvirally mediated gene transfer using a noninfectious virus to deliverthe gene in the patient or injecting naked DNA into a site in thepatient and the DNA is taken up by a percentage of cells in which thegene product protein is expressed. Additionally, the other methodsdescribed herein, such as use of a “gene gun,” may be used for in vitroinsertion of anti-angiogenic or anti-tumor polypeptide-encoding DNA andlinked regulatory sequences.

Chemical methods of gene therapy may involve a lipid based compound, notnecessarily a liposome, to transfer the DNA across the cell membrane.Lipofectins or cytofectins, lipid-based positive ions that bind tonegatively charged DNA, make a complex that can cross the cell membraneand allow the DNA into the interior of the cell. Another chemical methoduses receptor-based endocytosis, which involves binding a specificligand to a cell surface receptor and enveloping and transporting itacross the cell membrane. The ligand binds to the DNA and the wholecomplex is transported into the cell. The ligand gene complex isinjected into the bloodstream and then target cells that have thereceptor will specifically bind the ligand and transport the ligand-DNAcomplex into the cell.

A plasmid or other type of suitable vector can be constructed tofacilitate the expression of one or more gene sequences encoding aTSP-derived polypeptide as described herein. Strong promoter regions andenhancer regions may be used to facilitate the construction of such aplasmid. If the plasmid is not to be introduced directly at the site ofthe tumor cells to be treated, it maybe also be desirable to constructthe plasmid such that a segment of DNA encoding a signal peptide isinserted immediately 5′ to the coding region for the polypeptide.

Many gene therapy methodologies employ viral vectors to insert genesinto cells. For example, altered retrovirus vectors have been used in exvivo methods to introduce genes into peripheral and tumor-infiltratinglymphocytes, hepatocytes, epidermal cells, myocytes, or other somaticcells. These altered cells are then introduced into the patient toprovide the gene product from the inserted DNA.

Viral vectors have also been used to insert genes into cells using invivo protocols. To direct the tissue-specific expression of foreigngenes, cis-acting regulatory elements or promoters that are known to betissue-specific can be used. Alternatively, this can be achieved usingin situ delivery of DNA or viral vectors to specific anatomical sites invivo. For example, gene transfer to blood vessels in vivo was achievedby implanting in vitro transduced endothelial cells in chosen sites onarterial walls. The virus infected surrounding cells which alsoexpressed the gene product. A viral vector can be delivered directly tothe in vivo site, by a catheter for example, thus allowing only certainareas to be infected by the virus, and providing long-term, sitespecific gene expression. In vivo gene transfer using retrovirus vectorshas also been demonstrated in mammary tissue and hepatic tissue byinjection of the altered virus into blood vessels leading to the organs.

Viral vectors that have been used for gene therapy protocols include butare not limited to, retroviruses, other RNA viruses such as poliovirusor Sindbis virus, adenovirus, adeno-associated virus, herpes viruses,SV40, vaccinia and other DNA viruses. Replication-defective murineretroviral vectors have been widely utilized gene transfer vectors.

Carrier mediated gene transfer in vivo can be used to transfect foreignDNA into cells. The carrier-DNA complex can be conveniently introducedinto body fluids or the bloodstream and then site-specifically directedto the target organ or tissue in the body. Both liposomes andpolycations, such as polylysine, lipofectins or cytofectins, can beused. Liposomes can be developed which are cell specific or organspecific and thus the foreign DNA carried by the liposome will be takenup by target cells. Injection of immunoliposomes that are targeted to aspecific receptor on certain cells can be used as a convenient method ofinserting the DNA into the cells bearing the receptor. Another carriersystem that has been used is the asialoglycoprotein/polylysine conjugatesystem for carrying DNA to hepatocytes for in vivo gene transfer.

The gene therapy protocol for transfecting anti-tumor polypeptides intoa patient may either be through integration of a gene encoding apolypeptide into the genome of the cells, into minichromosomes or as aseparate replicating or non-replicating DNA construct in the cytoplasmor nucleoplasm of the cell. Anti-angiogenic polypeptide expression maycontinue for a long period of time or the carrier-DNA complex may bereinjected periodically to maintain a desired level of the polypeptidein the cell, the tissue or organ or a determined blood level.

Gene transfer into muscle cells especially has been tested and found tobe safe. In a recent test, biologically active endostatin was producedby expression plasmids injected into muscle cells, and inhibited tumorvascularization and the growth of subcutaneous tumors and metastaticlung tumors. (Blezinger, P. et al., Nature Biotechnology 17:343–348,1999). An appropriate dose of plasmid can be injected intramuscularly,for example, once weekly.

Peptide phage display libraries have been used to identify peptides thatbind specifically to the vascular endothelial cells of different organssuch as lung, skin, pancreas and intestine (Rajotte, D. et al., J. Clin.Invest., 102:430–437, 1998). The individual binding properties of thevasculature of specific organs can be used in targeting the delivery ofan agent, such as a polypeptide, to those vascular cells.

Peptides found to bind to the endothelial cells of capillaries supplyingblood to individual organs can be produced in fusion with theTSP-derived polypeptides of the invention, to provide a chemical addressto target a particular tissue type of organ. For example, the expressionvector described above can be engineered to have a coding sequence for atargeting peptide immediately 5′ or immediately 3′ of the codingsequence for a TSP-derived polypeptide of the invention. Such vectorscan be administered to a tumor-bearing mammal, such as a human, at thesite of one or more tumors, or at a distant site, to provide a source offusion polypeptide by expression of the fusion gene on the vector.Alternatively, a recombinant fusion protein of a targeting peptide and aTSP-derived polypeptide can be produced in host cells cultured in alaboratory or production facility, using an expression vector or usinghost cells otherwise genetically altered to produce the fusionpolypeptide.

Not only have peptides been found that bind specifically to the vascularendothelial cells of different organs, but peptides have been found thatbind specifically to molecules exposed on the surface of tumor cells andangiogenic tumor vasculature. See, for example, Burg, M. A. et al.,Cancer Research, 59:2869–2874, 1999, in which phage display of a peptidelibrary was used to select for phages binding to NG2 proteoglycan, whichis expressed on the surface of several different types of tumors.

EXAMPLES Example 1

Production and Characterization of Recombinant Polypeptides

Human TSP-1 and Recombinant Proteins.

Human TSP-1 was purified from the supernatant of thrombin-treatedplatelets as described previously (Adams, J. C. and Lawler, J, Mol. Bio.Cell 5: 423–437, 1994). Recombinant proteins that included sequencesfrom the type 1 repeats were prepared by PCR using the full-length cDNAfor human TSP-1 as a template. A DNA segment encoding a recombinantprotein containing all three TSRs of TSP-1 (3TSR, amino acids 361–530;SEQ ID NO: 20) was prepared using the forward primer 475htsp1f (GAT GATCCC GGG GAC GAC TCT GCG GAC GAT GGC; SEQ ID NO: 10) and the reverseprimer 476htsp1r (GAT ACC GGT AAT TGG ACA GTC CTG CTT G; SEQ ID NO: 11).A DNA segment encoding a recombinant protein that contains the secondTSR (TSR2, amino acids 416–473; SEQ ID NO: 21) was prepared using theforward primer 537htsp1f (GAT GAT CCC GGG CAG GAT GGT GGC TGG AGC; SEQID NO: 12) and the reverse primer 515htsp1r (GAT ACC GGT GAT GGG GCA GGCGTC TTT CTT; SEQ ID NO: 13). To evaluate the role of TGFβ activation onthe effect of this recombinant protein, a DNA segment encoding a longerversion of the second TSR (TSR2+RFK, amino acids 411–473; SEQ ID NO: 22)that includes the RFK sequence was synthesized using the forward primer514htsp1f (GAT GAT CCC GGG GAC AAG AGA TTT AAA CAG; SEQ ID NO: 14) andthe reverse primer 515htsp1r. All three PCR products were cloned betweenthe XmaI and the AgeI sites of the vector pMT/BiP/V5-HisA (Invitrogen,Carlsbad, Calif.). The recombinant proteins included the vector-derivedamino acid sequence RSPWG (SEQ ID NO: 15) at the NH₂-terminus andTGHHHHHH (SEQ ID NO: 16) at the COOH-terminus. The fidelity of the PCRproducts was verified by nucleotide sequencing. Each expression vectorwas cotransfected into Drosophila S2 cells with the selection vectorpCoHYGRO according to the manufacturer's protocols (Invitrogen).Transfected cells were selected with hygromycin B and the expression ofrecombinant polypeptides was monitored by western blotting using thepolyclonal antibody R3 that was raised against a fusion protein thatcontained all three TSRs of TSP-1 (Legrand, C. et al., Blood,79:1995–2003, 1992). For large-scale preparation of recombinant protein,S2 cells were grown in serum-free media for five days. The culturesupernatant was centrifuged to remove the cells and dialyzed against 20mM NaPO₄ (pH 7.8) and 500 mM NaCl. The dialysate was applied to a columnof ProBond resin (Invitrogen). The column was eluted with 20 mM NaPO₄(pH 6.0), 500 mM NaCl, 500 mM imidazole. The protein eluted with 500 mMimidazole was dialyzed against 20 mM NaPO₄ (pH 7.0), and 500 mM NaCl and1% sucrose was added prior to storage.

Cell Culture.

Human dermal microvessel endothelial cells (HDMEC) were isolated by theprocedure of Richard et al., Exp. Cell Res. 240:1–6 (1998). The cellswere cultured in Vitrogen precoated dishes, and maintained in EBM(Clonetics Corp, San Diego, Calif.) containing 20% fetal bovine serum(FBS), 1 mg/ml hydrocortisone acetate, 5×10⁻⁵ M dibutyryl-cAMP, 200 U/mlpenicillin, 100 U/ml streptomycin, 250 mg/ml amphotericin and 2–5 ng/mlVEGF. Murine melanoma B16F10 and murine Lewis lung carcinoma cells wereobtained from the ATCC, and were maintained in Dulbecco's Modified EagleMedia (DMEM) supplemented with 10% FBS, 50 mg/ml penicillin, 50 U/mlstreptomycin, and 2 mM glutamine (supplemented DMEM). Mink lungepithelial cells (MLECs-Clone 32) that have been stably transfected withan 800 bp fragment of the plasminogen activator inhibitor-1 (PAI-1)promoter fused to the firefly luciferase reporter gene were kindlyprovided by Dr. Daniel Rifkin (Abe, M. et al., Anal. Biochem.216:276–284, 1994). The transfected MLECs were maintained insupplemented DMEM.

Assay for TGFβ Activation.

B16F10 cells (2.5×10⁵) were plated in a T25 flask and grown overnight insupplemented DMEM. The cells were rinsed once with 1.0 ml of serum-freeDMEM and 2.5 ml of serum-free DMEM containing 5.0 mg/ml of the TSR, orTSR+RFK recombinant protein were added. After an overnight incubation,conditioned media was collected and centrifuged at 12,000 rpm for 5 minto remove cellular debris. Undiluted media was used to determine thelevel of active TGFβ. The level of total TGFβ was determined byincubating the media at 80° C. for 10 min and diluting 1:5 in DMEM. Onehundred microliters of conditioned media were added to wells of a96-well plate containing 3×10⁴ MLECs and the plates were incubatedovernight. A standard curve was constructed by incubating 7.8 to 250pg/ml of purified TGFβ with the MLECs® & D Systems, Inc. Minneapolis,Minn.) in DMEM. The media was removed and the cells were washed 3 timeswith cold PBS prior to the addition of 100 ml of lysis buffer(PharMingen, San Diego, Calif.). Luciferase activity was measured usingthe Enhanced Luciferase Assay Kit PharMingen).

In Vitro Migration Assay.

Cells at passage 7–10 were serum-starved and maintained in EBM with 0.1%BSA (control media) for 20 hours before trypsinization to harvest thecells. Cells were washed in EBM twice and resuspended in control mediaat a concentration of 1×10⁶ cells/ml. Two hundred ml of cells werepacked down and kept frozen. They were lysed and serially diluted byCyQuant reagent (Molecular Probe, Eugene, Oreg.) to be used for standardcurve construction.

Transwell membrane (24-well polycarbonate membrane, 8 mM pore size fromCorning Costar Corporation, Cambridge, Mass.) coated with Vitrogen (30mg/ml, Invitrogen) on both sides was used for chemotactic migrationexperiments. Coated transwells were inverted, 100 ml of cell suspensionapplied to the top of the membrane and covered by the bottom platecarefully so that the cell suspension stayed on top of the membrane. Thecells were allowed to adhere to the coated membrane for two hours in anincubator at 37° C. with 5% CO₂. After the adhesion incubation, theplates were re-inverted, the bottom wells were filled with 0.4 mlcontrol media and the top wells were filled with 0.1 ml control mediacontaining testing reagents. The plate was returned to the incubator for3.5 hours for the cells to migrate. At the end of the incubation, thetranswells were washed in PBS and the cells on the bottom side of themembrane (unmigrated cells) were wiped away with a cotton swab. Themembranes were cut out by scalpel and placed into 96-well plates andfrozen at −80° C. overnight. Two hundred ml of CyQuant reagent wereadded to each well. Fluorescence reading was done 16 hours later with aSpectraFluor plate reader with excitation at 485 nm and emission at 535μm. The number of cells migrated was then calculated based on thestandard curve.

Characterization of Recombinant Proteins

Three recombinant forms of the type 1 repeats of human TSP-1 have beenproduced in S2 cells. A particular example of a polypeptide comprisingthe three type 1 repeats of human TSP-1, but not comprising the otherdomains of TSP-1 designated “3TSR,” has been made, and contains aminoacids 361–530 of human TSP-1. This protein has a predicted molecularweight of 20,520 Daltons and an apparent molecular weight of 25,000Daltons on SDS-PAGE, suggesting that carbohydrates are added bypost-translational modification. The 3TSR, TSR+RFK and TSR recombinantproteins were electrophoresed on a 4%–15% gradient gel. The gel was cutin half and duplicate lanes were stained with Coomassie blue, or westernblotted with a polyclonal antibody to all three TSRs of human TSP-1.

Each TSR contains six cysteine residues. Consistent with the presence ofintrachain disulfide bonds in this protein, the 3TSR protein migratesmore rapidly in the absence of reducing agent during SDS-PAGE (Panetti,T. S. et al., J. Biol. Chem., 274:430–437, 1999). A second construct,designated TSR2+RFK, contains the second type 1 repeat of human TSP-1(amino acids 411–473; SEQ ID NO: 22) and includes the DKRFK (SEQ ID NO:2) sequence in the NH₂-terminal region. The RFK sequence has been shownto be necessary and sufficient for activation of TGFβ (Schultz-Cherry,S. et al., J. Biol. Chem., 270:7304–7310, 1995). An equivalent region ofTSP-1 (amino acids 416–473; SEQ ID NO: 21) that excludes the DKRFK (SEQID NO: 2) sequence, designated TSR2, has also been expressed. Theaverage level of protein expression is comparable for all three proteins(approximately 24 μg of purified protein from 1 ml of conditionedmedia). All three proteins react with a polyclonal antibody, designatedR3, that was raised against a bacterial fusion protein composed of thetype 1 repeats fused to β-galactosidase (Legrand, C. et al., Blood,79:1995–2003, 1992). Taken together, the data indicate that therecombinant proteins produced in S2 cells are similar to the type 1repeats in the native protein.

Functionally, the recombinant TSR-containing proteins are similar to thenative TSRs in that they activate TGFβ and inhibit endothelial cellmigration. In media conditioned by B16F10 melanoma cells, 6.3% of thetotal TGFβ is in the active form (FIG. 1). Addition of 5 μg/ml of theTSR2+RFK protein to conditioned media increases the level of the activeform to 30% of the total TGFβ. By contrast, addition of TSR2 does notresult in an increase in the level of active TGFβ in the conditionedmedia (4.8%). As shown in FIGS. 2A and 2B, TSP-1 is a potent inhibitorof endothelial cell migration in vitro. This inhibition isdose-dependent up to 100 ng/ml of TSP-1, but concentrations of TSP-1above this level are less effective. This biphasic response has beenreported by others (Tolsma, S. S. et al., J. Cell Biol., 122: 497–511,1993). The 3TSR, TSR2 and TSR2+RFK recombinant proteins also inhibitendothelial migration with responses that are similar to TSP-1, on aweight basis. All three recombinant proteins are maximally effective ininhibiting endothelial cell migration at 10 ng/ml (FIGS. 2A and 2B).

Example 2

Testing Effect of Recombinant Polypeptides on Tumor Growth

Primary Tumor Growth Assay.

The proteins for injection were mixed with Polymyxin B-Agarose (Sigma)for 30 minutes at room temperature to remove endotoxin. The endotoxinlevels were less than 0.05 EU/mg as determined using the QCL-1000 assaykit (Biowhittaker, Walkersville, Md.). Proteins were filter-sterilizedand the protein concentration was determined prior to injection.

Five to eight week old C57BL/6 mice (Taconic, Germantown, N.Y.) wereacclimated, caged in groups of four or less and their backs were shaved.Cultured B16F10 melanoma or Lewis lung carcinoma cells (1×10⁶) wereinoculated subcutaneously on the back of each mouse. Tumors weremeasured with a dial-caliper and the volumes were determined using theformula width²×length×0.52. The treatment began 4 days after inoculationof the tumor cells. The therapeutic groups received TSP-1 or recombinantTSP-1 proteins IP daily, and the negative control group receivedcomparable injections of saline alone. The experiments were terminatedand mice sacrificed when the control mice began to die, usually at day16 post-tumor cell injection.

Histological Examination.

The tumor tissues were cut, fixed with neutral buffered formaldehyde,and embedded in paraffin according to standard histological procedures.Hematoxylin and eosin staining was employed for tissue morphologyexamination. Blood vessels were immunochemically stained by anti-CD31antibody with a Vectastain ABC kit (Vector Laboratories, Burlingame,Calif.). Tumor cell apoptosis was detected by TUNEL assay (Abe, M. etal., Anal. Biochem, 216:276–84, 1994). The number of blood vessels wasrecorded by counting ten high-power fields. Tumor cell proliferation andthe apoptotic index were estimated by the percentage of cells scoredunder a light microscope. A minimum of 1,000 cells was counted in eachtumor sample.

Inhibition of Experimental Tumor Growth by Recombinant Proteins

The B16F10 and the Lewis lung carcinoma models of experimental tumorgrowth have been used to assay the effect of the recombinant proteins.These models have been used extensively to assay the activity of theanti-angiogenic proteins endostatin, angiostatin and anti-thrombin III(O'Reilly, M. S. et al. Science, 285:1926–8, 1999; O'Reilly, M. S. etal, Cell 88:277–85, 1997; Boehm, T. et al., Nature 390:404–407, 1997).Systemic injection of TSP-1 (0.25 mg/ml) into tumor-bearing miceinhibits tumor growth (FIG. 3A) by 56% on the twelfth treatment day. Athigher doses less inhibition is observed and a dose of 2.5 mg/kg/day hasno effect on tumor growth. The 3TSR protein is a less effectiveinhibitor of tumor growth at 0.25 mg/kg/day than the intact protein(FIG. 3B). By contrast, at dosages of 1.0 mg/kg/day and greater, the3TSR protein is more effective than TSP-1. Tumor volume is reduced by81% by the 3TSR protein with a dose of 2.5 mg/kg/day and it remainsmaximally active at 10 mg/kg/day.

The inhibitory effect of the 3TSR protein is also observed in micebearing experimental tumors that are formed by subcutaneous injection ofLewis lung carcinoma cells. At 2.5 mg/kg/day, the 3TSR protein inhibitstumor growth by 73% on treatment day 12 (FIG. 4). The TSR2+RFK proteinis equally as effective as the 3TSR protein on a weight basis. ForB16F10 and Lewis lung carcinoma cell-derived tumors, this proteininhibits tumor growth by 86% and 77%, respectively (FIGS. 4 and 5A).

To explore the potential involvement of the RFK sequence in theinhibition of B16F10 tumor growth, the TSR2+RFK, and TSR2 proteins, havebeen assayed. The tumor inhibition effect of the TSR2+RFK protein wascomparable to that of the 3TSR protein at the various doses used (FIGS.4B and 6A). By contrast, TSR2 is significantly less effective (FIG. 6B).At 0.25 mg/kg/day, the tumor growth in the mice treated with the TSR2protein was indistinguishable from the control group. At 1.0 mg/kg/day,the TSR2+RFK protein inhibited tumor growth by 80%, while the TSR2protein only inhibited growth by 38% on the twelfth day of treatment. Atthe highest dose tested (2.5 mg/kg/day), the difference was lesspronounced with the TSR2+RFK and TSR2 proteins inhibiting tumor growthby 86% and 68%, respectively.

To better understand the effect of the recombinant protein treatment ontumor growth, we have determined the rate of proliferation, theapoptotic index and the capillary density for tumors in the salinecontrol group, mice treated with the TSR2+RFK protein or mice treatedwith the TSR2 protein at an intermediate dose (1.0 mg/kg/day) where thelargest effect of inclusion of the RFK sequence is observed. Tumors wereremoved and fixed on the twelfth treatment day. Capillaries werevisualized with anti-CD31 antibody and apoptotic cells were identifiedby TUNEL. Tumors displayed a 71% and 64% reduction in capillary densitywhen the mice were treated with the TSR2+RFK and TSR2 proteins,respectively (FIG. 6A). Whereas the tumors from mice that were treatedwith the TSR2+RFK protein displayed a 4-fold increase in tumor cellapoptosis p<0.005), the tumors from the mice that were treated with theTSR2 protein displayed only a 1.9-fold increase in apoptosis (p≦0.025)(FIG. 6B). The TSR2+RFK protein also reduced the percentage of PCNApositive tumor cells. The number of proliferating cells was decreased by35% by treatment with the TSR2+RFK protein (p<0.005), while treatmentwith the TSR2 protein reduced tumor cell proliferation by 7.8% (p<0.05)(FIG. 6C).

Constructs for Expression of TSR2+QFK and 3TSR (TSP-2) Polypeptides

A recombinant protein in which arginine (413) was mutated to glutaminewas prepared using the forward primer 605 htsp1f (GAT GAT CCC GGG GACAAG CAA TTT AAA CAG GAT GG; SEQ ID NO: 17) and the reverse primer 515htsp1r (GAT ACC GGT GAT GGG GCA GGC GTC TTT CTT; SEQ ID NO: 13). Theresulting PCR product contains the glutamine codon CAA instead of thearginine codon AGA. The template for the PCR reaction was humanthrombospondin-1 cDNA and the cloning sites were XmaI and AgeI in thepMT/BiP/V5-HisA vector. This approach is equivalent to that used for theconstruction of TSR2+RFK.

This approach was also used to construct the recombinant protein withall three TSRs of mouse TSP-2. In this case, a full-length cDNA clonefor mouse TSP-2 was used as the template and the primers 544 mtsp2f (GATGAT CCC GGG GAT GAG GGC TGG TCT CCG; SEQ ID NO: 18) and 545 mtsp2r (GATACC GGT AAT AGG GCA GCT TCT CTT; SEQ ID NO: 19) were used. The 3TSR(TSP-2) polypeptide of mouse consists of amino acids Asp 381 through Ile550 (SEQ ID NO: 25) as shown in FIG. 10 (SEQ ID NO: 24). By similarmethods, a 3TSR (TSP2) polypeptide of human could be made. See, forexample, National Center for Biotechnology Information Accession No. NM003247, Homo sapiens thrombospondin 2 mRNA. A polypeptide produced bythese methods would consist of amino acids Glu 381 through Val 550.

Inhibition of B16F10 Experimental Tumor Growth by Various RecombinantProteins TSR2, TSR2+RFK and TSR2+QFK.

Each treatment group contained six five- to eight-week old C57BL/6 mice.Treatment was initiated four days after subcutaneous injection of 1×10⁶B16F10 melanoma cells. The mice received one IP injection each day ofsaline, TSR2, TSR2+RFK or TSR2+QFK (SEQ ID NO: 23). A soluble form ofthe TGFβ receptor (100 μg/mouse/injection) (Smith, J. D. et al.,Circulation Research 84:1212–1222, 1999) was included in the saline orTSR2+RFK injections on treatment day 1 and 7 for two additionalexperimental groups. The recombinant TSR-containing proteins were usedat 1.0 mg/kg/day. Results are shown in FIG. 8.

Inhibition of B16F10 Experimental Tumor Growth by All Three Type 1Repeats of Human TSP-1 or Mouse TSP-2.

Each treatment group contained six five- to eight-week old C57BL/6 mice.Treatment was initiated four days after subcutaneous injections of 1×10⁶B16F10 melanoma cells. The mice received one IP injection each day, ofsaline, all three type 1 repeats of human TSP-1 [3TSR (TSP1) in FIG. 9]or all three type 1 repeats of mouse TSP-2 [3TSR (TSP2) in FIG. 9].Recombinant proteins were used at a dose of 2.5 mg/kg/day.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A pharmaceutical composition comprising a polypeptide consisting ofSEQ ID NO: 22, or a variant of the polypeptide, wherein the variant hasat least about the same apoptotic or anti-angiogenic activity as thepolypeptide, and wherein the variant is at least 70% identical to thepolypeptide, and a pharmaceutically acceptable carrier.
 2. A polypeptideconsisting of amino acids 411–473 of human TSP-1 (SEQ ID NO: 22).
 3. Amethod of inhibiting growth of angiogenesis dependent tumors in apatient, comprising administering to said patient an amount of thepharmaceutical composition of claim 1 sufficient to inhibit the growthof the tumors.
 4. A method for inhibiting neovascularization inangiogenesis dependent tumors in a patient, said method comprisingadministering to the patient an amount of the pharmaceutical compositionof claim 1 sufficient to inhibit neovascularization in said tumors.
 5. Amethod of decreasing proliferation of angiogenesis dependent tumor cellsin a patient, comprising administering to said patient an amount of thepolypeptide of claim 2 sufficient to decrease proliferation of saidtumor cells.
 6. A method for inhibiting neovascularization in anangiogenesis dependent tumor or rumors in a patient, said methodcomprising administering to the patient an amount of the pharmaceuticalcomposition of claim 2 sufficient to inhibit neovascularization in thetumor or tumors.
 7. A polypeptide comprising the three type 1 repeats ofhuman TSP-1 (SEQ ID NO: 20), but not comprising other domains of TSP-1.8. A polypeptide comprising SEQ ID NO: 22, but not comprising otherdomains of TSP-1.
 9. A polypeptide comprising all three type 1 repeatsof human TSP-2 (SEQ ID NO: 27), but not other domains of TSP-2.
 10. Apharmaceutical composition comprising a human 3TSR (TSP2) polypeptide(SEQ ID NO. 27), and a pharmaceutically acceptable carrier.
 11. A methodfor reducing volume or inhibiting growth of an angiogenesis dependenttumor in a patient, comprising administering to the patient an amount ofthe polypeptide of claim 8 sufficient to reduce the volume of or inhibitthe growth of said tumor.
 12. A polypeptide consisting of SEQ ID NO: 20.13. A method of inhibiting the growth of an angiogenesis dependent tumorin a patient, said method comprising administering to the patient anamount of the polypeptide of claim 12 sufficient to inhibit the growthof said tumor.
 14. A method of inhibiting the growth of an angiogenesisdependent tumor in a patient, said method comprising administering tothe patient an amount of the polypeptide of claim 7 sufficient toinhibit the growth of said tumor.
 15. A method for inhibiting growth ofmelanoma tumors or lung tumors in a patient, said method comprisingadministering to the patient an amount of a polypeptide consisting ofSEQ ID NO: 22 or a variant of the polypeptide, wherein the variant hasat least about the same apoptotic or anti-angiogenic activity as thepolypeptide, and wherein the variant is at least 70% identical to thepolypeptide.
 16. A method for inhibiting growth of melanoma tumors orlung tumors in a patient, said method comprising administering to thepatient an amount of a polypeptide consisting of SEQ ID NO: 20 or avariant of the polypeptide, wherein the variant has at least about thesame apoptotic or anti-angiogenic activity as the polypeptide, andwherein the variant is at least 70% identical to the polypeptide.